Graduation Date

Summer 8-14-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Pharmaceutical Sciences

First Advisor

Tatiana K. Bronich

Abstract

Combination chemotherapy remains the mainstay of cancer treatment because such strategy targets different cell signaling pathways to decrease the likelihood of developing protective mechanisms by cancer cells, thereby delaying the onset of recurrence and prolonging the survival. The co-delivery of binary drug combination via a single nanocarrier provides benefits in reducing dose-limiting toxicities, improving the pharmacokinetic properties of the cargo, spatial-temporal synchronization of drug exposure, and synergistic therapeutic effects. Rational design of such regimen is crucial for maximizing the therapeutic effects since only certain drug ratios exposed to the target might be synergistic while other ratios exert additive or even antagonistic effects. Cisplatin-based chemotherapy has shown great responses in several cancer types and has been used as the standard systemic anticancer treatment, such as triple negative breast cancer (TNBC) and ovarian cancer. However, tumor cells are becoming less responsive to cisplatin, which arises from altered signaling patterns of cancer cells, such as activation of pro-survival signal transduction (e.g., epidermal growth factor receptor (EGFR), AKT, PI3-kinase) and DNA-damage repair, etc. EGFR is a member of the HER family of oncogenic receptor tyrosine kinases (RTK), which actively participates in sustaining the growth and the survival of carcinoma cells as well as the pathogenesis and progression of different carcinoma types. It is frequently overexpressed in various cancer types, such as TNBC and ovarian cancer, making it a major therapeutic target for the development of targeted drug delivery. A combination of EGFR inhibitors with cisplatin have shown strong synergistic effects in EGFR overexpressing cancers both in preclinical and clinical studies. Moreover, the combined regimen significantly outperformed the single treatment as reported. It has been shown preclinically that EGFR inhibitors significantly potentiate the cytotoxic effects of cisplatin in tumors with overexpressed EGFR through blocking oncogenic signal transduction and unfavored pro-survival signals induced by platinum-based therapy. However, inhibiting EGFR could trigger underappreciated resistance and activate the parallel oncogenic signaling pathways through other members of HER family. Using a pan-HER inhibitor is a common strategy to avoid this limitation. To this end, neratinib (NRT) (an FDA-approved pan-HER inhibitor) and cisplatin (CDDP) were selected as the combination regimen to treat EGFR+ cancers: TNBC and ovarian cancer. To incorporate these two molecules with varied physicochemical properties in the same carrier, we designed a biocompatible crosslinked polypeptide-based nanogel (NG) with multifunctional compartments. Such biodegradable platform provides flexibility in adjusting size, loading capacity, surface properties, deformability, softness, and responsive behaviors by tuning the chemical compositions as well as crosslinking levels. For the purpose of the current study, NGs prepared from copolymers poly (ethylene glycol)-block-poly (L-glutamic acid) modified with phenylalanine functionalities ((PEG-P(Glu-Glu/Phex)150) were developed for simultaneous loading and delivery of binary CDDP and NRT combination. Such NGs have 1) a hydrophilic PEG shell for less RES uptake and extended circulation, 2) an anionic crosslinked core, which incorporates CDDP through coordination with the carboxylic groups of PGlu, 3) hydrophobic regions formed by Phe moieties, which serves as a reservoir for NRT solubilization. By tuning ratio of constituent hydrophilic and hydrophobic moieties, NGs displayed varied dimensions, drug loading capacities, deformability, performance in killing tumor cells, as well as penetrations in multicellular 3D tumor models. Consistent with properties of chemical composition, NGs with high hydrophobic fraction displayed less swelling ability and more efficient hydrophobic drug loading. Resulting binary drug combination-loaded NGs functionalized with 50 units of hydrophobic Phe were able to encapsulate both CDDP and NRT at a molar ratio of 2:1 and displayed the strongest synergistic effect towards EGFR+ TNBC cells compared with other screened regimens. Such superior synergy was found to be selective and only displayed in the EGFR+ TNBC cell line. Notably, NRT was found to reverse pro-survival signal transduction by CDDP mediated EGFR/Akt/Erk activation as well as increased Cyclin D1 expression, which was believed to be the molecular basis for the synergistic effect of the combination of CDDP and NRT. Drug-loaded carrier system exerted the highest synergy was selected in the following studies targeting EGFR overexpressing ovarian cancer. However, this carrier system solely depends on the EPR effect for drug accumulation in tumor sites, which is limited by heterogeneity among tumor masses as well as vascular density nearby influencing the permeability of nanocarriers. Installing targeting ligands on the surface of carriers to specifically target biomarkers overexpressed on the surface of tumor cells is a well-suited strategy to overcome mentioned limitations and increase retention of payloads in tumors. In the next part of our study, two targeting ligands aiming to bind with EGFR was chosen in our study: L-AE peptide and EGFR-A protein with reported high binding specificity and affinity to EGFR with acceptable stability. Two types of ligand-installed NGs both demonstrated success in significantly increased cellular uptake in EGFR+ ovarian cancer cells as compared to nontargeted NGs. Further optimization was conducted by extending the thickness of PEG stealth layer from 114 monomer units to 228 units for sufficient protection from opsonization. Optimized PEG-based polymeric NGs displayed more favored PK properties, such as remarkably less spleen uptake, higher drug retention in tumor sites, slower clearance from circulation, and more drug exposure. NGs with EGFR targeting ligands further improved the PK profile by directing remarkably more drugs to the target sites. When tested in vivo, EGFR targeted peptide and protein decorated NGs carrying CDDP and NRT drug combination significantly suppressed the growth of intraperitoneal high-grade serous ovarian tumor xenografts outperforming their nontargeted counterparts without extending their cytotoxicity to the normal tissues. We also confirmed the importance of simultaneous administration of the (CDDP+NRT) via a single NG system which provides more therapeutic benefits than a cocktail of individual drug-loaded NGs administered at equivalent doses. Lastly, our data demonstrated the benefits of local treatment by showing that intraperitoneal (IP) administration of targeted binary drug combination-loaded NGs can be more effective in terms of tumor growth suppression. These data have shown the power of our carrier system in the delivery of a drug combination to treat EGFR overexpressing cancers.

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